Methods for treating neuropathological states and neurogenic inflammatory states and methods for identifying compounds useful therein

ABSTRACT

The present invention provides methods for treating neuropathological states and neurogenic inflammatory states in a subject. The present invention also provides methods for identifying compounds that can be used to treat such states. Preferably, the compounds alter the distribution of NMDA glutamate receptor NR1 subunit in cells, and/or alter the production of TNFα by cells.

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Ser.No. 60/210,413, filed Jun. 8, 2000, and U.S. Provisional ApplicationSer. No. 60/225,702, filed Aug. 16, 2000, which are incorporated byreference herein.

GOVERNMENT FUNDING

The present invention was made with government support under Grant No.NS32778 and NS11255, awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

BACKGROUND

Glutamate is the primary transmitter of most neurons in the nervoussystem, including those involved in sensing pain. Glutamate is typicallylocked up in nerve endings and is released in very small amounts atnerve junctions to signal when a nerve is activated. Glutamate attachesitself to specific receptive docking sites on the next nerve insequence. In the case of ionotropic glutamate receptors the dockingprocess allows a tiny channel to open, and a charge is transferred bysodium and calcium ions as a tiny electrical current. In the case ofpain, the chemical message is converted to electrical energy and carriedby the pain transmission nerves to specific sites in the nervous systemthat interpret the signals as pain. Typically, the precision of thenervous system depends on this event taking place in microseconds. Theglutamate receptor site then closes rapidly, resetting for the nextneuronal event.

Glutamate is released by neurons in high concentrations in each case ofpersistent pain and neural injury. In cases of severe pain,inflammation, and tissue damage, such as with arthritis, spinal cordinjury or head injury, large amounts of glutamate escape and can bedestructive. The damage that occurs in the presence of high glutamateconcentrations translates into long-lasting pathological levels of painand nervous tissue damage. These events also involve many other neuronaland inflammatory agents, but excess glutamate is an initiator in theselong-term events that enhances the pain signal. In addition to prolongedpain, excessive glutamate for extended periods of time, as in severeinjury, will poison and kill nerve cells. This is evident in cases ofhead injury and spinal cord damage where the secondary destruction bythe presence of excess glutamate amplifies the initial damage caused bythe event itself.

Nerve activation under normal conditions involves a glutamate receptor,non-NMDA ionotropic glutamate receptor, allowing sodium to enter thecell to activate the cell. Another glutamate receptor, NMDA ionotropicglutamate receptor, is a channel on the cell membrane allowing passageof calcium ions that carries a stronger electrical signal in normaltransmission. This particular glutamate receptor, the NMDA glutamatereceptor, is composed of two sets of two protein molecules, the NR1 andNR2 NMDA glutamate receptor subunits. They bind tightly to one anotherto form the ring through which the calcium signal is carried. A fifthsubunit protein may sometimes accompany the four functional subunits.

In the case of persistent pain, the continual presence of glutamate willinitiate a cascade of additional events beyond simple nervous eventsignaling. In addition to activation of both ion channel type glutamatereceptors, other types of glutamate receptor complexes are activatedcalled metabotropic receptor proteins. These are receptor proteins thatwhen also activated have longer-lasting effects, sit adjacent to theNMDA glutamate receptor and can release stores of calcium inside thecell. Cascades of intracellular processes are then initiated. Mostimportantly, these cascades are capable of influencing the entirebehavior of the nerve cell in a longer-lasting way if they signal anddirect the future activities of the cell long-term by communicating withthe cell nucleus.

Memory and learning function are closely related to persistent painmechanisms. Memory and learning involve both types of ion transportingglutamate receptors, the ionotropic glutamate receptors, in thehippocampus and cortex of the brain. The cells in the hippocampus areactivated by glutamate as the signal relaying transmitter. If theactivation of this brain region is strong enough and persistent enough,then both ionotropic glutamate receptor types are activated to achieve along-term memory of the event through a new “hard-wired” corticalneuronal circuit. This is similar to the “hard-wired” memory of thepainful event that becomes a persistent process in the pain transmissioncircuitry, referred to as sensitization on the cellular level andpersistent central pain state on the whole animal level. Furtheractivation of the event to the point of becoming pathological amounts ofchronic pain occurs by activation of other neuronal receptors,preferably in this case the metabotropic glutamate receptor producinglong-term nuclear and cellular changes including the release ofintracellular calcium and regulation of many enzymes (especially kinasesand phosphatases). The cells are then overactivated and a sensitized,neuropathological state develops. In the neuropathological state thepain message is enhanced and may remain this way for a prolonged periodof time.

The aim of basic science research in the field of pain is to providebetter treatment for the millions of people who suffer from persistentpain. Current methods for pain control mainly use traditionalpharmacological approaches in which small molecules are taken orally.Many of these medicines treat some of the symptoms of pain by attachingthemselves to receptor molecules on the cell surface, including theglutamate receptor, in an attempt to compete with the nerves owntransmitter chemicals. Currently, little is known about the processesthat have already occurred within the cell that are already initiatedafter neuropathological neural signaling events have occurred and bywhich the high degree of persistent pain continues. A betterunderstanding of these events would allow for more effective paincontrol.

SUMMARY OF THE INVENTION

This invention represents a significant advance in the art ofidentifying agents that can be used for the treatment of certainconditions, including neuropathological states and neurogenicinflammatory states.

The present invention provides a method for treating a neuropathologicalstate in a subject. The method includes administering to the subject aneffective amount of a tyrosine kinase inhibitor. The neuropathologicalstate may be, for instance, persistent pain, arthritis, ulcerativecolitis, inflammatory bowel disease, Crohn's disease, pancreatitis,asthma, stroke, brain injury, spinal cord injury, epileptogenesis, orviral invasion. The tyrosine kinase inhibitor may be, for instance,Genistein, Lavendustin A, K252a, or combinations thereof.

In another aspect, a method for treating a neuropathological state in asubject includes administering to the subject an effective amount of acompound that decreases the amount of NR1 subunit associated with anucleus of a cell of the subject wherein the cell contains an NMDAglutamate receptor. The cell may be a neuron. Optionally, the neuron isa sensitized neuron or is prevented from being converted to a sensitizedneuron. The compound may be a tyrosine kinase inhibitor such asGenistein, Lavendustin A, K252a, or combinations thereof.

In a further aspect, a method for treating a neuropathological state ina subject includes administering to the subject an effective amount of acompound that decreases the amount of Tumor Necrosis Factor alpha (TNFα)produced by a cell of the subject. The cell may be a synovial cell. Thecell may optionally include an NMDA glutamate receptor. The compound maybe a tyrosine kinase inhibitor such as Genistein, Lavendustin A, K252a,or combinations thereof.

The present invention also provides a method for identifying a compoundthat alters NR1 subunit distribution in a cell. The method includescontacting a cell with an effective amount of the compound, activatingan NMDA glutamate receptor present on the cell, and detecting thedistribution of NR1 subunit in the cell wherein detection of analteration in the distribution of NR1 subunit in the cell contacted withthe compound relative to the distribution of NR1 subunit in a cell notcontacted with the compound indicates an alteration in the distributionof NR1 subunit. The cell may be a neuron. The compound may be a tyrosinekinase inhibitor.

The present invention provides a method for identifying a compound thatalters the production of TNFα by a cell. The method includes contactinga cell with an effective amount of the compound, activating an NMDAglutamate receptor present on the cell, and detecting the amount of TNFαproduced by the cell wherein detection of an alteration in the amount ofTNFα produced by the cell contacted with the compound relative to theamount of TNFα produced by a cell not contacted with the compoundindicates an alteration in the amount of TNFα produced by the cell. Thecell may be a synovial cell.

The present invention provides a method for treating a neurogenicinflammatory state in a subject. The method includes administering tothe subject an effective amount of a compound that decreases the amountof TNFα produced by a cell of the subject wherein the cell contains anNMDA glutamate receptor. The compound may be a tyrosine kinaseinhibitor.

The present invention provides a method for treating arthritis in asubject. The method includes administering to the subject an effectiveamount of a compound that decreases the amount of TNFα produced by asynovial cell of the subject. The compound may be a tyrosine kinaseinhibitor.

The present invention provides a method for altering NR1 subunitdistribution in a cell. The method includes contacting a cell with aneffective amount of the compound, activating an NMDA glutamate receptorpresent in the cell, and detecting the distribution of NR1 subunit inthe cell wherein detection of an alteration in the distribution of NR1subunit in the cell contacted with the compound relative to thedistribution of NR1 subunit in a cell not contacted with compoundindicates an alteration in the distribution of NR1 subunit. The NR1subunit associated with a nucleus of a cell of the subject may beincreased or decreased.

Definitions

As used herein, the terms “neuropathological state” and“neuropathological condition” are used interchangeably and refer tofunctional disturbances and/or pathologic changes in a subject's nervoussystem. Examples of functional disturbances include persistent pain, aninflammatory state, brain injury, spinal cord injury, epileptogenesis,and viral invasion. Examples of pathological changes include thepresence of persistent pain due to functional disturbances and adecrease in mental function due to epileptogenesis, memory disturbances,and aging.

As used herein, “neurogenic inflammatory state” refers to conditionswhere high concentrations of a glutamate receptor agonist are present(for instance, released by a neuron) and interact with a glutamatereceptor, preferably an NMDA glutamate receptor, that is present on acell. The interaction of the agonist with the glutamate receptor resultsin the production of cytokines, preferably, tumor necrosis factor α(TNFα) by the cell. Types of neurogenic inflammatory states include, forinstance, arthritis, ulcerative colitis, inflammatory bowel disease,Crohn's disease, pancreatitis, asthma, stroke, brain injury, and viralinvasion.

As used herein, an “effective amount” is an amount effective to decreaseor prevent in a subject the symptoms associated with a conditiondescribed herein.

As used herein, the term “glutamate receptor” refers to a receptorpresent on the outer cellular membrane of a cell that binds glutamate,glutamate agonists, or glutamate antagonists. There are two types ofglutamate receptors, ionotropic and metabotropic. Binding of agonist,for instance glutamate, N-methyl-D-aspartate, aspartate (NMDA), glycine,serine, by an ionotropic glutamate receptor results in channel openingand the subsequent flow of ions through the channel. Binding of agonist,for instance glutamate, (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylicacid (ACPD), quisqualic acid or ibotenic acid by a metabotropicglutamate receptor results in phosphoinositide hydrolysis andintracellular calcium mobilization. “Activation” of a transmitter-gatedchannel, for instance a glutamate receptor, refers to the opening of thechannel or initiation of transmitter related events. Preferably, theglutamate receptor is an NMDA glutamate receptor.

As used herein, a “tyrosine kinase inhibitor” is a compound thatinhibits the activity of a tyrosine kinase to catalyze the transfer of aphosphate group, typically the terminal phosphate group from anadenosine triphosphate (ATP) molecule, to a tyrosine residue present ina target protein. A tyrosine kinase inhibitor can act on receptorshaving intrinsic tyrosine kinase activity (receptor tyrosine kinases)and tyrosine kinases that are not associated with a receptor(non-receptor protein tyrosine kinases). Preferably, a tyrosine kinaseinhibitor acts on a non-receptor protein tyrosine kinase.

As used herein, unless otherwise noted, the terms “translocate” and“translocation” refer to movement of the NR1 subunit from the cellmembrane to the nuclear membrane, preferably the inner nuclear membrane.

As used herein, the term “neuron” refers to a conducting cell of thenervous system. A neuron releases a neurotransmitter, preferablyglutamate, that binds a transmitter-gated channel, preferably aglutamate receptor, located on the cell surface of a cell. The cellhaving the glutamate receptor, preferably an NMDA glutamate receptor,may be a neuron or other cells as described herein.

As used herein, the term “subject” includes humans, as well as otheranimals (for instance, mice, rats, or rabbits) that can be used asanimal models in the study of the conditions described herein.

As used herein, the term “NR1 subunit” refers to the NMDA glutamatereceptor NR1 subunit.

As used herein, the term “sensitized neuron” refers to a neuron that hasbeen altered such that activation of the neuron results in a responsethat is greatly enhanced relative to a non-sensitized neuron. Sensitizedneurons play a role in allodynia or hyperalgesia. As used herein, theterm “allodynia” refers to an increased sensitivity to a stimulus thatwas previously innocuous. For instance, a stimulus that was previouslyinnocuous is now considered painful, i.e., noxious. As used herein, theterm “hyperalgesia” refers to an increased sensitivity to a noxiousstimulus. Allodynia and hyperalgesia can be primary or secondary.Primary allodynia and primary hyperalgesia mean the location of theincreased sensitivity is at the same site as an injury. Secondaryallodynia and secondary hyperalgesia mean the location of the increasedsensitivity is at a site that is not identical as the site of theinjury. A sensitized neuron typically has higher amounts of proteinkinase C and nitric oxide (NO) synthase than neurons that are notsensitized.

As used herein, the term “persistent pain” refers to pain that continuesfor at least about 10 minutes after the initial stimulus causing thepain. Chronic pain is pain that lasts at least 3 weeks. Persistent painincludes chronic pain and any type of pain that lasts at least about 10minutes.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Paw withdrawal latency (PWL) versus time. Base, beginning ofexperiment; 1.5 h, 1.5 hours; 4 h, 4 hours; drug, time at whichLavendustin A, Genistein, Daidzein, Lavendustin B, or vehicle wasadministered as described herein; k/c, time at which kaolin/carrageenanwas administered as described herein to produce the experimentalarthritis; *, one way Analysis of Variance (ANOVA) repeated measurement,planned comparison, p<0.01 versus baseline; #, one way ANOVAbetween-groups, planned comparison, p<0.05 (versus 4 hour Lavendustin Agroup); ˆ, one way ANOVA between-groups, planned comparison, p<0.01(versus 4 hour vehicle A group).

FIG. 2. The effect of Genistein pre-treatment on NR1 stain density inthe superficial dorsal horn (I-II) of the spinal cord in rats. Normal,density of receptors in the superficial dorsal horn (I-II) of the spinalcord in rats not treated; vehicle (dimethyl sulfoxide, DMSO)+Arthritisdensity of receptors in the superficial dorsal horn (I-II) of the spinalcord in rats treated with DMSO 1.5 hours before treatment of the kneejoint with the irritant kaolin/carrageenan; Genistein+Arthritis, densityof receptors in the superficial dorsal horn (I-II) of the spinal cord inrats treated with the tyrosine kinase inhibitor, Genistein 1.5 hoursbefore treatment with kaolin/carrageenan; *, t-test for independentsamples, p<0.05 (versus normal); NR1, NMDA glutamate receptor NR1subunit; phospho-R1, antibody for phosphatase activated NMDA glutamatereceptor NR1 subunit; NR1 A/B, NMDA glutamate receptor NR1 subunitsubtypes A/B; NR2C, NMDA glutamate receptor NR2 subunit subtype C;mGluR1, group I metabotropic glutamate receptor; mGluR5, Group 5metabotropic glutamate receptor; n, number of animals.

FIG. 3. Western blot analysis detecting the presence of the glutamateNR1 receptor subunit in cytosolic and nuclear extracts from culturedclonal human synovial cells SW982 treated with increasing concentrationsof NMDA.

FIG. 4. Tumor necrosis factor-alpha (TNF-α) levels in the culture mediumof clonal human synovial cells SW982 treated with NMDA and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ACPD (N+A) in the presenceof the tyrosine kinase inhibitors Genistein (G) or K252a (K). N=3independent replicas per group. # and *: p<0.01 vs. control orNA-treated cells, respectively (unpaired Student's “t” test), pg/ml,picograms per milliliter.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides methods for identifying compounds thatalter the distribution of NR1 subunits and/or alter the production ofTumor Necrosis Factor Alpha (TNFα) in a cell. The cell can be ex vivo orin vivo. As used herein, the term “ex vivo” refers to a cell that hasbeen removed from the body of a subject. Ex vivo cells include, forinstance, primary cells (e.g., cells that have recently been removedfrom a subject and are capable of limited growth in tissue culturemedium), and cultured cells (e.g., cells that are capable of extendedculture in tissue culture medium). As used herein, the term “in vivo”refers to a cell that is within the body of a subject. Preferably, thecell is ex vivo.

Cells useful in the present invention have a glutamate receptor,preferably an NMDA glutamate receptor, on the cell surface. Examples ofsuch cells include, for instance, neurons. Examples of useful neuronsthat can be used ex vivo include cultured neuroblastoma cells,preferably rat, mouse, or human, more preferably human. An example of acultured human neuroblastoma cell is SHSY5Y (ATCC CRL 2266). Otherexamples of useful ex vivo neurons include neurons isolated from thedorsal horn of the spinal cord, dorsal root ganglia and other cellbodies of peripheral nerves, hippocampal or other limbic or corticalneurons. Preferably the neurons are removed from a rat. Examples of invivo neurons include neurons in the spinal cord, for instance neurons inthe dorsal horn and motor horn, the brain, for instance neurons in thebasal forebrain and hippocampus, peripheral neurons, for instance dorsalroot ganglia.

Examples of other cells useful in the present invention include, forinstance, cultured synovial sarcoma cells, preferably rat, mouse, orhuman, more preferably human. An example of a cultured human synovialsarcoma cell is SW982 (ATCC HBT-93). Examples of in vivo cells includesynovial cells lining a knee joint of a subject, preferably a human.

In an aspect of the present invention, the methods include evaluatingthe effect of different compounds by contacting a cell with a compound,activating a glutamate receptor, preferably an NMDA glutamate receptor,present on the cell, and detecting the distribution of the NR1 subunitin the cell. The distribution of the NR1 subunit in the cell is comparedto the distribution in a cell that was not exposed to the compound.Contacting the cell with a compound can occur before, during, or afteractivating a glutamate receptor present in the cell. Cells ex vivo canbe contacted directly with the compound by, for instance, adding thecompound to the media in which the cell is bathed. Cells in vivo can becontacted directly with a compound. For instance, cells of the spinalcord can be contacted directly as described in Example 1. Alternatively,a compound can be introduced to the animal systemically as apharmaceutical composition. Pharmaceutical compositions are detailedherein.

In another aspect of the present invention, the methods includeevaluating the effect of different compounds by contacting a cell with acompound, activating a glutamate receptor, preferably an NMDA glutamatereceptor, present on the cell, and detecting the production of TNFα bythe cell. The amount of TNFα produced by the cell is compared to theamount of TNFα produced by a cell that was not exposed to the compound.Contacting the cell with a compound can occur before, during, or afteractivating a glutamate receptor present in the cell. Cells ex vivo canbe contacted directly with the compound by, for instance, adding thecompound to the media in which the cell is bathed. Cells in vivo can becontacted directly with a compound. Alternatively, a compound can beintroduced to the animal systemically as a pharmaceutical composition.

The invention is not intended to be limited by the types of compoundsthat can be screened for activity using the methods described herein.Accordingly, a compound can be, for instance, a polypeptide, an organicmolecule, polyketide, or a non-ribosomal peptide. Compounds useful inthe methods of the present invention can be produced by naturalorganisms, or produced using methods known to the art including, forinstance, recombinant techniques, or chemical or enzymatic synthesistechniques. Preferably, the compound is not produced by a mammal.

Preferred examples of compounds that can be used in some aspects of thepresent invention include tyrosine kinase inhibitors. Tyrosine kinaseinhibitors are known in the art and include, for instance, Genistein(5,7-Dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one;4′,5,7-trihydroxy-isoflavone, Catalog Number G-103 from RBI, Natick,Mass.), Lavendustin A(5-Amino-[N-2,5-dihydroxybenzyl)-N′-2-hydroxybenzyl]salicylic acid,Catalog Number 428150 from Calbiochem, La Jolla, Calif.), and K252a(Catalog Number 420298, from Calbiochem, La Jolla, Calif.). Whether acompound is a tyrosine kinase inhibitor can be determined using methodsknown in the art (see, for instance, Akiyama et al., J. Biol. Chem.,262, 5592-5595 (1987) and Omichi et al., Biochemistry, 31(16:4034-4039(1992)). Preferably, a tyrosine kinase inhibitor useful in the presentinvention decreases phosphorylation of NR1 receptor. Without beinglimiting, it is expected that a specific tyrosine kinase mediates thetranslocation of NR1 subunit, and that this specific tyrosine kinasewill be a member of one of the known families of tyrosine kinases, forinstance, the Src or Jak families. Inhibitors are available thatspecifically inhibit individual members of the known families oftyrosine kinases. Accordingly, it is expected that specific tyrosinekinase inhibitors will be useful in the methods of the presentinvention. Alternatively, it is expected that other useful compoundsinclude tyrosine phosphatases and serine/threonine phosphatases. Inother aspects of the present invention, preferred examples of compoundsthat can be used include tyrosine kinases, tyrosine phosphataseinhibitors, or serine/threonine phosphatase inhibitors where increasesin NR1 subunit would be advantageous such as for improving memory orslowing the aging process.

The amount of a compound that is administered to alter the distributionof NR1 subunits in a cell, or alter the production of TNFα by a cell,varies depending on the type of compound used. Typically, when acompound is screened for activity in the methods of the presentinvention, various concentrations of the compound are used.

Activation of a neuron ex vivo can be accomplished by incubating a cellin tissue culture media and adding at least about 5 micromolar of aglutamate receptor agonist, and more preferably at least about 10micromolar of a glutamate receptor agonist. Typically, translocation ofNR1 subunit to the nuclear membrane can be observed about 4-24 hoursafter exposure. Neurogenic inflammatory states can be experimentallyrecreated ex vivo by adding at least 5 micromolar, preferably at leastabout 10 micromolar, of a glutamate receptor agonist to a cell thatincludes a glutamate receptor, and measuring the resulting production ofTNFα by the cells. Alterations in the amount of TNFα produced by a cellcan be observed about 4-24 hours after exposure.

Activation of a cell in vivo is typically accomplished by using ananimal model that can be used for investigating conditions that resultfrom sensitization of cells or from increased concentrations ofglutamate. Without intending to be limiting, such conditions includeneuropathological states and neurogenic inflammatory states. Animalmodels for studying these conditions are known in the art and can beused in the methods of the present invention. Models for the study ofpain include those approved by the International Association for theStudy of Pain. A preferred animal model for identifying compounds thatalter the distribution of NR1 subunits in a cell is the rat arthritismodel described in Example 1. The rat arthritis model is a commonlyaccepted model for the study of pain and arthritis in humans. Otheranimal models (for instance, using cat, monkey, or rabbit as the animal)are also commonly accepted models for these human conditions (see, e.g.,Neugebaurer & Schaible, Agents and Actions, 25, 234-236 (1988) andO'Byrne et al., Arthritis and Rheumatism, 33, 1023-1028 (1990)). Tostudy pain in this model, cells in the spinal cord are exposed to acompound for a period of time, and then a knee of the animal is exposedto a stimulus that evokes persistent pain. Methods of evoking persistentpain are known in the art. After a period of time the responsiveness ofthe animal to an innocuous or noxious stimulus is evaluated usingmethods known in the art. A compound that causes an animal to havereduced primary allodynia or secondary allodynia, or reduced primaryhyperplasia or secondary hyperplasia compared to an animal that has notreceived the compound indicates that the distribution of NR1 subunits inthe spinal cord is altered. This model may also be used to studyarthritis. A compound can be injected into the synovial space of a kneejoint, and then the knee exposed to a stimulus that evokes arthritis.After a period of time, the responsiveness of the animal to movements inthe working range of the joint are evaluated. A compound that causes ananimal to have reduced response time when its foot is touched, that is,the animal overreacts, compared to an animal that has not received thecompound indicates that the distribution of NR1 subunits in the cellslining the synovial space, and/or the production of TNFα by the cells isaltered. Neuropathological states and neurogenic inflammatory states canalso be experimentally recreated in vivo by injecting a glutamatereceptor agonist into a space containing neurons (for instance, thespinal cord) or other cells (for instance, the synovial space present ina joint) that include a glutamate receptor.

The distribution of the NR1 subunits present in a cell exposed to thecompound is measured and compared to a cell that has not been exposed tothe compound. The distribution of NR1 subunits present in a cell can bemeasured using methods known in the art for determining the location ofa polypeptide in a cell. For instance, the amount of NR1 subunitsassociated with the cellular membrane, present in the cytoplasm, orassociated with the nucleus can be determined. Preferably, the amount ofNR1 subunits associated with the nucleus, more preferably associatedwith the nuclear membrane, most preferably associated with the innernuclear membrane is determined. The amount of NR1 subunit associatedwith the nucleus can be increased or decreased, preferably decreased, ina cell contacted with a compound.

Typically, the presence of NR1 is assayed using NR1-specific antibodiesand methods known to the art including western immunoblot,immunoprecipitation, and immunocytochemistry. Without intending to belimiting, for example, cells can be fractionated and the differentsubcellular fractions tested for the presence of NR1 subunits.Alternatively, cells can be fixed and sectioned for analysis by, forinstance, immunocytochemical analysis, using light microscopy orelectron microscopy.

Alternatively, instead of detecting alterations in the distribution ofNR1 subunits in the cell, the total amount of NR1 present in a cellcontacted with a compound can be determined and compared to the totalamount of NR1 present in a cell not contacted with a compound. The smallamount of NR1 subunit in the nucleus normally is at the level ofdetection while an increase can be measured five hours after inductionof knee joint inflammation. Methods for determining the total amount ofa polypeptide in a cell or cell fractions are known in the art. Inanother alternative, instead of detecting alterations in thedistribution of NR1 subunits in the cell, the amount of phosphorylatedNR1 subunit present in a cell contacted with a compound can bedetermined and compared to the amount of the activated form of thesubunit, phosphorylated NR1 present in a cell not contacted with acompound. Methods for determining whether a polypeptide isphosphorylated are known in the art.

Due to the observed presence of NR1 subunits in the nucleus, it isexpected that NR1 subunits alter gene expression. Accordingly, it isexpected that changes in gene expression, including alterations intranscription or translation, preferably translation, may also be usedto measure alterations in the distribution of NR1 subunits in a cell.

The production of TNFα by a cell can be measured using methods known inthe art, including, for instance, Enzyme-linked Immunosorbant Assay (BLISA), and ex vivo biological assays. The TNFα can be TNFα presentinside the cell, secreted by the cell, or a combination thereof.Preferably, the TNFα measured is TNFα that has been secreted by thecell. Preferably, the amount of TNFα produced by a cell contacted with acompound is decreased.

The present invention is further directed to methods for treatingcertain conditions in a subject. The conditions include, for instance, aneuropathological state such as persistent pain, stroke, brain injury,spinal cord injury, epileptogenesis, and viral invasion, and neurogenicinflammation such as arthritis, stroke, ulcerative colitis, inflammatorybowel disease, Crohn's disease, pancreatitis, asthma, spinal cordinjury, and viral invasion.

The methods include administering to the subject an effective amount ofa compound that decreases or prevents a symptom of a neuropathologicalstate or neurogenic inflammatory state. The compounds useful in thisaspect of the invention are described above and can be used alone or incombination. Preferably, the compound is a tyrosine kinase inhibitor,more preferably Genistein, Lavendustin A, or K252a. The subject can bean animal, preferably a rat, a mouse, or a human, most preferably ahuman.

Treatment described herein can be prophylactic (initiated before asubject manifests symptoms of a condition described herein) or,alternatively, can be initiated after the development of a conditiondescribed herein. Accordingly, administration of a compound can beperformed before, during, or after the occurrence of the conditionsdescribed herein. Treatment initiated after the development of acondition may result in decreasing the severity of a symptom of thecondition, or completely removing a symptom of the condition. Thecompound can be administered systemically. When administeredsystemically, the compound is preferably associated with an agent thatwill direct the compound to the appropriate cells. For example, thecompound can be associated with an antibody to a receptor present on thesurface of a cell such that the compound is transported to the interiorof the cell, for instance by endocytosis. Preferably, the receptor isendocytosed through clathrin-coated pits to endosomes. The compound canbe administered locally. In the treatment of persistent pain thatresults from cancer or back pain the compound can be administered by,for instance, intraspinal catheter. In the treatment of arthritis thecompound can be administered by, for instance, injection of the compoundinto the synovial space of the affected joint. Preferably, the compoundis administered locally.

An aspect of the invention is directed to a method for treating aneuropathological state in a subject. The method includes administeringto the subject an effective amount of a compound, preferably a tyrosinekinase inhibitor, more preferably Genistein, Lavendustin A, or K252a.

In some aspects of the invention, the compound used to treat the subjectalters the distribution of NR1 subunit in a cell, preferably decreasesthe amount of NR1 subunit associated with the nucleus of a cell.Alternatively, the compound decreases the total amount of NR1 in thecell, or decreases the amount of phosphorylated NR1 in the cell.Preferably, the compound both decreases the amount of NR1 subunitassociated with a cell's nucleus and decreases the total amount of NR1in the cell. In other aspects of the invention, the compound used totreat the subject alters the production of TNFα by a cell, preferablydecreases the production of TNFα by the cell. In some aspects, thecompound may both alter the distribution of NR1 in a cell and alter theproduction of TNFα by the cell.

A neural cell can be present in the spinal cord, for instance in thedorsal horn, in the brain, for instance in the basal forebrain orhippocampus, or in the cell body of a peripheral neuron, for instance adorsal root ganglia. Other glutamate receptor-containing cells of thepresent invention can be present, for instance, in the knee joint suchas synovial cells. In aspects of the invention that are directed todecreasing a symptom of a neuropathological state, preferably the cellis a sensitized neuron. It is expected that decreasing a symptom of aneuropathological state results in converting a neuron from a sensitizedstate to a non-sensitized state. In aspects of the invention that aredirected to preventing a symptom of a neuropathological state,preferably the cell is a non-sensitized neuron. It is expected thatpreventing a symptom of a neuropathological state results in preventingthe conversion of a cell from a non-sensitized state to a sensitizedstate.

Also provided by the present invention are methods for treating aneurogenic inflammatory state in a subject. The method includesadministering to the subject an effective amount of a compound,preferably a tyrosine kinase inhibitor, more preferably Genistein,Lavendustin A, or K252a.

In the case of a neurogenic inflammatory state of a subject caused bynon-neuronal cells producing TNFα in response to a glutamate receptoragonist (for instance, arthritis, ulcerative colitis, inflammatory boweldisease, Crohn's disease, pancreatitis, asthma, stroke, brain injury,and viral invasion), it is expected that decreasing a symptom of aneurogenic inflammatory state will result in decreasing or preventingcells from producing TNFα. Without intending to be limiting, it isexpected that, by reducing TNFα production, the capacity of TNFα tostimulate the inflammatory cascade is reduced, thereby decreasing,preferably preventing, a neurogenic inflammatory state.

In the case of a neurogenic inflammatory state located in the nervoussystem of a subject, such as neurogenic inflammation occurring during aneuropathological state of a subject (for instance, stroke, spinal cordinjury, and viral invasion), TNFα expression and/or secretion from thecell is also reduced, thereby decreasing, preferably preventing,neurogenic inflammation occurring during a neuropathological state. Forinstance, a subject suffering from a severe spinal cord injury maydevelop a neuropathological state as well as a neurogenic inflammatorystate. Under these circumstances, administration of a compound of thepresent invention is capable of decreasing, preferably preventing, theneuropathological state and the neurogenic inflammatory state.

The present invention provides methods for altering the ability of asubject to retain information, e.g., increasing the memory of a subject.The method includes administering to the subject an effective amount ofa compound that increases the ability of a subject to retaininformation. Whether a subject is able to retain information can bedetermined using methods known to the art. For instance, when thismethod is used with non-humans, maze tests can be used, and when thismethod is used with humans, tests such as the California learning scaleor the Wisconsin memory test can be used. Preferably, short-term memoryor long-term memory is increased, more preferably, long-term memory isincreased.

The compounds useful in this aspect of the invention are describedabove. Preferably, the compound is a tyrosine kinase, a tyrosinephosphatase inhibitor, a serine/threonine phosphatase inhibitor, orcombinations thereof. Typically, the compound alters the distribution ofNR1 subunit in a neuron or cells targeted by neurons, preferably byincreases the amount of NR1 subunit associated with the nucleus of aneuron or cell. Alternatively, the compound increases the total amountof NR1 in the neuron or target cell, or increases the amount ofphosphorylated NR1 in the neuron or target cell. Preferably, thecompound both increases the amount of NR1 subunit associated with thenucleus of a neuron and increases the total amount of NR1 in the neuron.The subject can be an animal, preferably a rat, a mouse, or a human,most preferably a human. The neuron is typically present in the brain,preferably in the hippocampus.

The compound(s) useful in the methods disclosed herein is optionally andpreferably present in a pharmaceutically acceptable carrier. Thecompounds useful in the present invention, preferably a tyrosine kinaseinhibitor, may be formulated in pharmaceutical preparations in a varietyof forms adapted to the chosen route of administration. Formulationsinclude those suitable for parental administration (for instanceintramuscular, intraperitoneal, intraspinal catheter, intrasynovial, orintravenous), oral, transdermal, or nasal administration. Dosages of thecompositions of the invention are typically from about 0.1 mg/kg up toabout 50 mg/kg intravenous or 50-250 mg/kg oral dose. Preferably, anintraspinal dose is about 10 microliters of a solution containing fromabout 01 micromolar to about 1 micromolar of the solution.

The formulations may be conveniently presented in unit dosage form andmay be prepared by methods well known in the art of pharmacy. Allmethods of preparing a pharmaceutical composition include the step ofbringing the active compound (e.g., a tyrosine kinase inhibitor) intoassociation with a carrier that constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into the desired formulations.

Typically, the compositions of the invention will be administered fromabout 1 to about 4 times per day. The amount of active ingredient thatmay be combined with the carrier materials to produce a single dosageform will vary depending upon the subject treated and the particularmode of administration. A typical preparation will contain from about 5%to about 95% active compound (w/w). Preferably, such preparationscontain from about 20% to about 80% active compound. The amount ofcompound in such therapeutically useful compositions is such that thedosage level will be effective to prevent or suppress theneuropathological state or the neurogenic inflammatory state.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the composition, ordispersions of sterile powders that include the composition, which arepreferably isotonic with the blood or synovial fluid of the recipient.Isotonic agents that can be included in the liquid preparation includesugars, buffers, and sodium chloride. Solutions of the composition canbe prepared in water, and optionally mixed with a nontoxic surfactant.Dispersions of the composition can be prepared in water, ethanol, apolyol (such as glycerol, propylene glycol, liquid polyethylene glycols,and the like), vegetable oils, glycerol esters, and mixtures thereof.The ultimate dosage form is sterile, fluid, and stable under theconditions of manufacture and storage. The necessary fluidity can beachieved, for example, by using liposomes, by employing the appropriateparticle size in the case of dispersions, or by using surfactants.Sterilization of a liquid preparation can be achieved by any convenientmethod that preserves the bioactivity of the composition, preferably byfilter sterilization. Preferred methods for preparing powders includevacuum drying and freeze drying of the sterile injectable solutions.Subsequent microbial contamination can be prevented using variousantimicrobial agents, for example, antibacterial, antiviral andantifungal agents including parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. Absorption of the composition by theanimal over a prolonged period can be achieved by including, forexample, aluminum monostearate or gelatin.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as tablets, troches, capsules,lozenges, wafers, or cachets, each containing a predetermined amount ofthe active compound as a powder or granules, as liposomes containing theactive compound, or as a solution or suspension in an aqueous liquor ornon-aqueous liquid such as a syrup, an elixir, an emulsion or a draught.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: a binder such as gum tragacanth, acacia, cornstarch, or gelatin; an excipient such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; a sweetening agentsuch as sucrose, fructose, lactose or aspartame; and a natural orartificial flavoring agent. When the unit dosage form is a capsule, itmay further contain a liquid carrier, such as a vegetable oil or apolyethylene glycol. Various other materials may be present as coatingsor to otherwise modify the physical form of the solid unit dosage form.For instance, tablets, pills, or capsules may be coated with gelatin,wax, shellac, or sugar and the like. A syrup or elixir may contain oneor more of a sweetening agent, a preservative such as methyl orpropylparaben, an agent to retard crystallization of the sugar, an agentto increase the solubility of any other ingredient, such as a polyhydricalcohol, for example glycerol or sorbitol, a dye, and flavoring agent.The material used in preparing any unit dosage form is substantiallynontoxic in the amounts employed. The compound may be incorporated intosustained-release preparations and devices.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLE 1

Methods

Animals

All studies followed the guidelines of the Institute Animal Care and UseCommittee, in accordance with the guidelines of the National Institutesof Health. All animals were hosted in a room with a constant ambienttemperature of 22° C. and 12 hour light/dark cycle with free access tofood and water. Eighty-five Sprague-Dawley rats (250-300 grams) totalwere used in experiments conducted for biochemical, behavioral, andimmunocytochemistry studies. On Day 1, anesthetized animals receivedsurgical implantation of a microdialysis fiber for spinal administrationof tyrosine kinase inhibitors and inactive analogues. On Day 2 baselinebehavioral testing was followed by infusion of agents for 1.5 hours(pre-treatment) prior to induction of knee joint inflammation underbrief anesthesia. Behavioral testing was repeated 4 hours afterinduction of joint inflammation. Anesthetized animals were eithertranscardially perfused with aldehydes for immunohistochemical studiesor fresh, frozen tissues collected for biochemical studies.

Tyrosine Kinase Inhibitors

A microdialysis fiber was implanted into the spinal dorsal horn ofanesthetized rats (sodium pentobarbital, 50 mg/kg) one day prior to theinduction of arthritis according to the procedure of Skilling et al. (J.Neurochem., 51:127-132 (1988)), as described in Sluka et al. (Neurosci.Lett., 145:141-144 (1992)). Briefly, a small midline incision was madein the skin over the T12 vertebral level. The vertebrae were cleared ofmuscle and two 1 millimeter (mm) holes were drilled in the lateralaspect of both sides of the T12 vertebrae to expose the L4 spinalsegment. A microdialysis fiber (200 mm outer diameter, 45,000 MWcut-off, Hospal, AN69, Hospal Industrie, Meyzieu, France), was passedtransversely across the deep dorsal horn and stabilized with dentalcement. The microdialysis fiber was then passed through the holes in thevertebrae and transversely through the dorsal horn of the spinal cord. A2 mm section of the microdialysis fiber lies in the dorsal horn at thelevel of L3-4 segment. The tubing was coated with epoxy except for apermeable portion passing through the spinal grey matter. Themicrodialysis fiber was connected to PE₂₀ tubing (Becton Dickinson andCompany, San Mateo, Calif.) which was tunneled under the skin to thenape of the neck. Alternatively as a systemic control for drugadministration, the microdialysis fiber was implanted only in thesubcutaneous tissue over the back muscle and tunneled to the neck.Artificial cerebrospinal fluid (aCSF) was made as described by Sorkin etal. (J. Neurosci. Methods, 23, 131-138 (1988)) and was infused (5microliters/minute) for 1.5 hours and then the tube was heat-sealed foruse on the following day.

Two tyrosine kinase inhibitors and their inactive analogues werecompared in these behavioral studies. Genistein is a protein tyrosinekinase (PTK) inhibitor that decreases NMDA currents in patch clampstudies (Wang et al., Nature, 369, 233-235 (1994)). Daidzein is ananalogue of Genistein that lacks PTK inhibitory activity and has noeffect on NMDA currents in patch clamp studies (Wang et al., Nature,369, 233-235 (1994)). Lavendustin A, which is a structurally distinctPTK inhibitor that reversibly depresses NMDA currents, was also testedalong with its inactive analogue, Lavendustin B. The drugs (Genistein,Lavendustin A & B and Daidzein) were dissolved in 50 percent (%)dimethyl sulfoxide (DMSO) in aCSF. Dose response curves were generatedfor Genistein with animals receiving Genistein spinally at concentrationof 0.2 mM (n=3), 0.5 mM (n=3), 1 mM (n=9), and 2 mM (n=3). Animalsreceived Lavendustin A or B spinally at concentrations of 0.1 mM (n=3),0.5 mM (n=3), and 1 mM (n=5) as a positive and negative control forGenistein. The most effective dose in these pre-treatment animals was0.5 mM. Daidzein was tested at a dose of 1 mM (n=3). The most effectivedose for Genistein in these pre-treatment animals, 1 mM, was used forthe systemic control animals (n=3) receiving the drug subcutaneously.Eight additional animals received the vehicle, 50% DMSO in aCSF, as acontrol.

Based on in vitro estimates for Genistein measured by spectrophotometer(Beckman DU®650, Beckman Coulter, Inc., Fullerton, Calif.), a maximum ofabout 5.6% of the drug is transferred across the microdialysis membranemeasurable by high pressure liquid chromatography. Therefore, with thediffusion barriers presented by the tissue, the neurons are likely to beexposed maximally to a dose of Genistein (<56 micromolar) (μM) muchlower than that inside the microdialysis fiber (1 mM). The neurons arelikely to be exposed to a dose of Lavendustin (25 μM), a dose much lowerthan that inside the microdialysis fiber (0.5 mM).

Cycloheximide (100 milligrams per kilogram (mg/kg) intraperitoneal(i.p.) was used in some studies to determine if the alterations observedfor the NMDA NR1 receptor subunit could be attributed to newlysynthesized NR1 protein.

Induction of the Knee Joint Inflammation Model

a. Acute induction with kaolin/carrageenan. An acute inflammatoryresponse restricted to the knee joint can be induced by the injection of3% kaolin and 3% carrageenan (in sterile saline; 0.1 ml; pH 7.4) intothe joint cavity while the animal is briefly anesthetized with sodiummethohexital (Brevital, 60 mg/kg, i.p.). Kaolin and carrageenan wereobtained from Fisher Scientific, St. Louis, Mo. The knee joint is flexedmanually until the rat awakes (approximately 5-10 minutes). In thisarthritis model in the awake rat, localized joint swelling, as well aslimping and guarding of the limb, are well developed at 4 hours (Slukaet al., Pain, 59, 95-100 (1993)) when behavioral testing begins.

b. Measurement of Knee Joint Circumference. Knee joint circumference ismeasured in centimeters (cm) with a flexible tape measure around thecenter of the knee joint while the joint is held in extension as donewith patients in the clinical setting as an indication of increases injoint volume. The knee joints of anesthetized rats are measured before(baseline) and four hours following injection of the knee joint withkaolin and carrageenan.

c. Comparative Measurement of Joint Temperature. Temperature readingswere documented numerically with a temperature probe after the rat isre-anesthetized with sodium pentobarbital (50 mg/kg, i.p.) just prior toperfusion with aldehydes for histological preservation. Comparativetemperature differences were made between the baseline temperature andthe temperature four hours after induction of inflammation.

Behavioral Assessment

Fifty-five rats were used for behavioral studies. Four hours afterinduction of the arthritis, the joint is swollen and increasedwithdrawal responses to radiant heat and spontaneous guarding of thelimb were noted. The increased responsiveness to noxious stimuliindicates the presence of secondary hyperalgesia. Testing of pawwithdrawal latency (PWL) to radiant heat on the footpad using theHargreaves method (Hargreaves et al., Pain, 32, 77-88 (1988)), as ameasure of secondary hyperalgesia (away from the primary site of injuryindicative of central pain) reveals that the acute inflammation rendersthe hindlimb more sensitive to heat stimuli (Sluka et al., Pain, 59,95-100 (1994a)). Briefly, animals are placed in small Lucite cubicles ona glass top table cooled with a fan and allowed to accommodate for 30minutes prior to testing. A hand-held metal box focusing a highintensity light through an aperture (1 cm×0.8 cm) is used to applyradiant heat through the glass to the plantar surface of the hindpawuntil the animal lifts its foot. Radiant heat was applied to the plantarsurface of the hindpaw until the rat lifted its paw. The time which ittook for this to occur was considered the PWL response time. Both pawswere tested independently at five minute intervals for a total of fivetrials. A mean of these five reading was used as PWL response for eachtime point. Testing was done by the same observer for each test, and theobserver was blinded to the test groups under study. A decrease in PWLoccurred on the side ipsilateral to the inflamed knee 4 hours after theinduction of acute arthritis and was linearly correlated with theincrease in joint swelling. In the experimental rats the PWL wasmeasured before administration of drug or vehicle (baseline) and afterthe drug or vehicle had been infused for 1.5 hours at which time kaolinand carrageenan was injected into the knee joint. The final measurementfor PWL was at 4 hours after induction of arthritis. A decrease of thePWL to noxious radiant heat in a rat with knee joint inflammation isindicative of secondary hyperalgesia.

Immunocytochemical Localization of NMDA Receptor Subunit NR1

Twenty-four rats in 3 separate groups were used for immunocytochemicalstudies. One group of rat (n=8) was a naïve control. The other 2 groups(n=16) of rats received surgical placement of the microdialysis fiberplacement. On the day after microdialysis fiber placement, the animalswere treated spinally with either Genistein (1 mM) or vehicle (50% DMSOin aCSF) for 1.5 hours (n=8 for each group). Then the left knee joint ofall sixteen rats was injected with 0.1 ml of kaolin and carrageenan.

Animals were anesthetized and transcardially perfused with a brief warmsaline rinse followed by fixative solution (4% paraformaldehyde in 0.1 Mphosphate buffer, pH 7.4). The tissues were soaked overnight in 30%buffered sucrose and cut at 30 microns on a sliding microtome. For lightmicroscopy, the tissues from animals (n=8) were permeabilized with a 50%buffered ethanol treatment. Tissue sections were then stainedimmunocytochemically for glutamate receptor subunit NR1 and phospho-NR1.Primary antibodies to be used for staining include anti-NMDA NR1 NMDAreceptor (0.5-1 milligrams per milliliter (mg/ml)) purchased fromChemlcon Inc. (Pittsburgh, Pa.). Other neurotransmitter and receptorprimary antibodies against NMDA NR2C, mGluR1, and mGluR5 were used asstain specificity controls. The primary antibodies were diluted inphosphate buffered saline (PBS, pH 7.6) with 0.1% BSA for overnightincubation on the spinal cord tissues. After washing in PBS, the tissuesections were incubated in the appropriate secondary antibody, eitheranti-mouse or anti-rabbit IgG (1:100, 30 minutes) and in avidin-biotincomplex (ABC, 1:100, Vector Laboratories, Burlingame, Calif.). Sectionswere reacted with diaminobenzidine (DAB) solution (1.5 mg/ml) as thechromogen and peroxide (0.5 mg/ml) to produce a dense reaction product.To further intensify the reaction product particularly for visualizationof nuclear rings, some tissues were reacted with colloidal goldconjugated IgG and reacted with a silver chloride solution to produce anintense black reaction product (Takizawa et al., J. Histochemistry &Cytochemistry, 42, 1615-1623 (1994)).

Immunocytochemical controls included sections processed in the absenceof the primary or secondary antibody or ABC reagents. The specificity ofthe transmitter antibodies was confirmed by appearance of a single bandby Western blot. In addition to use of the C-terminus NR1 antibody fromChemIcon, an antibody directed to the N-terminus of the NR1 protein wasused as control (1 mg/ml; BD PharMingen, San Diego, Calif.).

For electron microscopy, the animals (n=3) were perfused with a mixtureof 2.5% glutaraldehyde and 1% paraformaldehyde. The lumbar cords werecut at a 30 mm thickness with a vibratome. After pretreatment with 1%sodium borohydride and the 50% buffered ethanol, tissue sections wereprocessed for immunocytochemical staining for mGluR1 and mGluR2/3 asabove. Tissues were dehydrated in alcohols, embedded in plastic resin,hardened and thin sectioned for electron microscopy.

Results

Behavioral Studies

Reflexive withdrawal of the paw (PWL) to radiant heat is known to bereduced from baseline 4 hours after induction of knee joint inflammationin this arthritis model in rats (Sluka et al., Neurosci. Lett.,145:141-144 (1993)). Comparisons were made with baseline and betweentreatment groups at 4 hours after joint inflammation (ANOVA). Thebehavioral studies demonstrated that pre-treatment with the proteintyrosine kinase inhibitor, Genistein, significantly attenuates theinflammation-induced decrease in PWL in response to radiant heatindicative of secondary hyperalgesia and a central sensitization statein the central nervous system in this arthritis model. Secondaryhyperalgesia requires sensitization of various portions of the neuronalcircuitry in addition to the local spinal reflex loop. Secondaryhyperalgesia which can be measured is a manifestation of centralsensitization. Thus central sensitization is sensitization of structuresof the pain circuitry as opposed to sensitization of the peripheralnerve of the spinal loop.

As shown in FIG. 1, Genistein and Lavendustin A significantly (p<0.05versus 4 hours vehicle group) attenuated the PWL decreases induced byknee joint inflammation in all treatment groups. Genistein (1 mM) andother agents tested did not affect baseline PWL. The structurallydistinct PTK inhibitor, Lavendustin A, also significantly abrogated thedevelopment of secondary hyperalgesia. While the secondary hyperalgesiadid not develop when Genistein and Lavendustin A were administered,other inflammatory signs were evident including the expected increasesin joint circumference and temperature.

Secondary hyperalgesia typical of this model developed afteradministration of vehicle and the inactive analogues, Daidzein andLavendustin B. Thus, control treatment with inactive analogs,Lavendustin B and Daidzein, were ineffective in altering the nociceptiveoutcome of knee joint inflammation. Secondary hyperalgesia is acentrally mediated form of nociceptive sensitization typical of thekaolin/carrageenan knee joint and other pain models. Since the tyrosinekinase inhibitor was applied directly to the affected spinal cordsegment by microdialysis, this confirms the requirement of tyrosinephosphorylation in long-term plastic changes occurring in the spinalcord that result in the development of secondary hyperalgesia. Whileongoing peripheral increases in joint temperature and circumference wereunaffected by this treatment, the nociceptive changes are not presentafter inhibition of protein tyrosine kinase.

Immunocytochemical Localization of Glutamate NMDA Receptor Subunit NR1

The density of immunocytochemical staining for NMDA NR1 in the lumbarenlargement on the side of the inflamed knee joint at 4 hours wasgreatly increased after induction of knee joint inflammation. Theincreases were evident as a diffuse stain density increase throughoutthe spinal grey matter. Computer-assisted measurement ofimmunocytochemical staining at the site of greatest increase in lamina Iand II indicated that stain density for NR1 was doubled in normalcontrols (FIG. 2). This increase was significant (P<0.05). Other NMDAreceptor subunits tested, including NR2A/B, NR2C, mGluR1, and mGluR5 didnot increase after knee joint inflammation. Likewise, an antibody to thephosphorylated form of NR1 did not increase after induction of kneejoint inflammation. In fact, at 4 hours after induction of the arthritismodel, the staining density for the phosphorylated form of NR1 wassignificantly decreased compared to normal control animals. The rapidincrease in NR1 staining also included a shift in the localizationpattern within the cell.

Significant quantities of NMDA NR1 were localized along the nuclearmembrane in animals with inflamed knee joints, forming a ring pattern atlight level (FIG. 2). With electron microscopy it was evident that thelocalization of NMDA NR1 was located on the inner nuclear membraneespecially at nuclear pore sites where post-transcriptionalmodifications can occur (FIG. 3).

After pre-treatment with Genistein, staining for NR1 was about half thatobserved in arthritic rats and was not significantly increased abovelevels seen in normal rats (FIG. 2). Likewise, the nuclear localizationwas greatly reduced. Staining was identical with specific antibodiesdirected against either the C- or N-terminus of the NR1 receptorsubunit. Pre-treatment with cycloheximide to eliminate de novo synthesisreduced the increase in NR1 staining by about half.

After induction of inflammation, the NMDA receptor subunit, NR1, is theonly NMDA receptor subunit tested that increased. The increasedexpression of NR1 occurred throughout the grey matter of the spinal cordon the same side as the knee joint inflammation. Genistein treatedarthritic animals had greatly reduced expression increases for NR1 thatwere not significantly different from baseline. Genistein also inhibitedthe shift in the pattern of NMDA NR1 protein staining from a lightdiffuse staining previously shown with electron microscopy to representpost-synaptic membrane localization (Liu et al., Proc. Natl. Acad. Sci.,91:8383-8387 (1994)) to that evident as nuclear translocation to theinner nuclear membrane shown here with electron microscopy. Thelocalization forms a ring pattern at the nuclear membrane notable atlight level within 4 hours after induction of arthritis. Thus, thenuclear translocation event is mediated by protein tyrosine kinasephosphorylation. Since cycloheximide reduced these increases it islikely that some of the increases in NR1 including the nuclearlocalization, represent newly synthesized NR1. Cycloheximide, whichprevents protein synthesis, did not totally reduce the staining tobaseline suggesting that some of the NR1 is translocating to the nucleusas well (see also results of Western blot analysis below).

The Role of NMDA Receptor NR1 Subunit Nuclear Translocation

The nuclear translocation of NMDA NR1 at nuclear pore sites suggests theability of the NMDA NR1 subunit to function at the nuclear membrane innuclear trafficking or perhaps to assist in other calcium-mediatedevents if dimers are formed. This signal transduction event requirestyrosine kinase phosphorylation. These results indicate that the NR1subunit is involved in glutamate mediated intracellular signalingpathways leading to central sensitization after nociceptive activation.

EXAMPLE 2

This example demonstrates that the translocation of NR1 subunit that wasobserved in animals also occurs in cultured cell lines.

Ex Vivo Studies

The cultured cell lines used in these studies were the humanneuroblastoma clonal line, SHSY5Y, and the human synovial sarcoma clonalline, SW982, both available through the American Tissue Type CultureCollection (item number CRL 2266 and HTB-93, respectively). Cells weregrown in Dulbecco's Modification of Eagles Medium (DMEM) withoutL-glutamine, with 4.5 grams/Liter glucose, and with 10% bovine fetalserum in a tissue culture incubator at 37° C. in a gaseous mixture ofO₂/CO₂. Cells were routinely split as necessary to maintain proper celldensity. Cells were removed from plates by adding TRYPSIN/EDTA in Hank'sBalanced Salt solution. After cell counts, approximately 10,000 cellswere plated per well in a culture dish. For experiments, the cells wereplated into 35 mm Petri dishes or 6- or 24-well plates as needed.

Cell Culture Treatments

For immunocytochemistry experiments and some western blot experiments,SW982 cells were exposed to glutamate (1 μM to 10 μM), or glutamateanalog NMDA (or related glutamate compound)(5 μM) and Genistein (10 mM).At various timepoints of exposure, including 4, 12, and 24 hours, cellswere collected for Western blot measurement of NR1 or fixed withaldehydes and stained to visualize the location of glutamate receptorsubunit NR1 using a specific antibody directed to either the C-terminusor the N-terminus of the NR1 protein.

For detection of released Tumor Necrosis Factor alpha (TNFα) and somewestern blot experiments, cells were exposed to the two glutamateagonists NMDA (5 mM) and ACPD (5 mM), in the presence or absence of thetyrosine kinase inhibitors Genistein (10 mM) or K252a (100 nM). After 24hours the culture medium was collected and processed for TNFα detection(see below) while the cells were lysed into cytosolic and nuclearprotein fractions and processed for western blot analysis as describedalso below.

Immunocytochemistry

Immunohistochemistry was used to identify glutamate receptor subtypespresent on cultured synoviocytes and neuroblastoma cells. Cells aretreated with fixative solution (4% paraformaldehyde in 0.1 M phosphatebuffer, pH 7.4) for 1 hour. Cultures are rinsed thoroughly (6 times)with phosphate buffered saline (PBS) with 0.1% BSA. Primary antibodiesused for staining include anti-NMDA NR1 glutamate receptor purchasedfrom Chemicon (Temecula, Calif. Catalog Number AB1516) (0.5-1 mg/ml)which will bind with the C-terminus of the ionotropic NMDA glutamatereceptor NR1 subunit specifically and the anti-NMDA NR1 antibody fromPharmingen (San Diego, Calif. Catalog Number 60021A) (1 mg/ml) whichbinds to the N-terminus of the NMDA glutamate receptor NR1 copy subunitspecifically. Other neurotransmitter and receptor primary antibodies arealso available in the lab for use as positive controls includingantibodies for the metabotropic glutamate receptors (anti-mGluR1 CatalogNumber 06-310, and anti-mGluR5 Catalog Number 06-451 from UpstateBiotechnology located in Lake Placid, N.Y.) and are used in dilutions of1:1,000-1:10,000 (adjusted empirically). The primary antibodies werediluted in phosphate buffered saline (PBS) with 0.1% BSA for overnightincubation on the spinal cord tissues. After washing in PBS (pH 7.6) thetissue sections were incubated in the secondary antibody, goatanti-rabbit IgG with a fluorescent ALEXA tag (1:100, 30 minutes)(Molecular Probes, Eugene, Oreg., Catalog Number A-11012).Immunocytochemical controls included sections processed in the absenceof the primary or secondary antibody. The specificity of the transmitterantibodies were confirmed by adsorption controls with excess antigen.Specific binding with antibodies identifying both the C- and theN-terminus, one of which was a monoclonal antibody, confirmed theidentity of the NR1 protein localization in the nucleus.

Western Blot Analysis

Western blot analysis was used to measure NR1 in both nuclear andcytosolic or total protein extracts using established methods (Kaufmannet al., J. Neurochem., 76, 1099-1108 (2001)). Extractions (20 μg ofprotein from nuclear/cytosolic fractions or 20-40 μg of protein fromtotal cell fractions) were diluted in 4×SDS loading buffer (each 100 mlof buffer contains: 3 grams (g) Tris; 8 g SDS; 2.5 g DTT; 0.05 gBromophenol blue; 40% [v:v] glycerol) and loaded onto a 10%SDS-polyacrylamide denaturing gel. After electrophoresis (1-2 hours at50 milliamps (mA)), the gel was blotted onto nitrocellulose membrane byovernight electrophoretic transfer. After blocking for 30 minutes atroom temperature with Tris-buffered saline containing 5% milk, membraneswere then incubated with a human NR1 rabbit polyclonal antibody(dilution 1:1,000 v:v, Chemicon, Temecula, Calif., Catalog Number ABI516) in Tris-buffered saline containing 2.5% powdered milk for 1 hour atroom temperature. Membranes were washed twice with Tris-buffered salineand then for 1 hour at room temperature in Tris-buffered salinecontaining 2.5% powdered milk with an HRP-conjugated goat anti-rabbitantibody (dilution 1:7,500 v:v, Biorad, Hercules, Calif., Catalog Number170/6515). After three washes with Tris-buffered saline, immunoreactivebands were detected by a chemiluminescent Western blot detection kit(Amersham, Buckingamshire, UK, Catalog Number RPN2106) according to themanufacturer's instructions.

Enzyme-Linked Immunosorbant Assay (ELISA)

The release of the inflammatory mediator and initiator of inflammatorycascades, TNFα was measured by ELISA using a commercially available kit(R&D Quantikine, Catalog Number DTA50) specific for human TNFα. TheSW982 cell culture media was removed as described above and centrifugedat 800×g for 10 minutes to remove floating cells and debris. Thesupernatant was then used to measure released TNFα using the R&D ELISAkit (DTA 50, Berkeley, Calif.) according to the manufacturer'sinstructions.

Results

Immunocytochemistry for clonal lines in culture treated with NMDA orNMDA plus a metabotropic glutamate receptor agonist replicated thevisualization of NR1 translocation to the nucleus. This was observed forboth the neuroblastoma and synovial clonal cell models, indicating thatthis is a general phenomenon of all cells and is not limited to thenervous system.

Western blot analysis allowed measurement of NR1 subunit either as atotal, nuclear, or cytosolic content. FIG. 3 shows results fromexperiments where it was determined by western blot analysis thepresence of the NR1 subunit in purified cytosolic and nuclear extractsfrom cultured human clonal synovial cells treated for 12 hours withincreasing concentrations of NMDA. A dose-dependent reduction of NR1 inthe cytosol was accompanied by the appearance of NR1 in the nuclear

1. A method for treating a neuropathological state in a subject, themethod comprising: administering to the subject an effective amount of atyrosine kinase inhibitor.
 2. The method of claim 1 wherein the tyrosinekinase inhibitor is selected from the group consisting of Genistein,Lavendustin A, K252a, and a combination thereof.
 3. The method of claim1 wherein the neuropathological state comprises a condition selectedfrom the group consisting of persistent pain, arthritis, ulcerativecolitis, inflammatory bowel disease, Crohn's disease, pancreatitis,asthma, stroke, brain injury, spinal cord injury, epileptogenesis, andviral invasion.
 4. (canceled)
 5. A method for treating aneuropathological state in a subject, the method comprising:administering to the subject an effective amount of a compound thatdecreases the amount of NR1 subunit associated with a nucleus of a cellof the subject, wherein the cell comprises an NMDA glutamate receptor.6. The method of claim 5 wherein the cell is a neuron.
 7. The method ofclaim 5 wherein the neuron is a sensitized neuron.
 8. The method ofclaim 5 wherein the neuron is prevented from being converted to asensitized state.
 9. The method of claim 5 wherein the neuropathologicalstate comprises a condition selected from the group consisting ofpersistent pain, arthritis, ulcerative colitis, inflammatory boweldisease, Crohn's disease, pancreatitis, asthma, stroke, brain injury,spinal cord injury, epileptogenesis, and viral invasion.
 10. The methodof claim 5 where in the compound is a tyrosine kinase inhibitor.
 11. Themethod of claim 10 wherein the tyrosine kinase inhibitor is selectedfrom the group consisting of Genistein, Lavendustin A, K252a, and acombination thereof.
 12. (canceled)
 13. A method for treating aneuropathological state in a subject, the method comprising:administering to the subject an effective amount of a compound thatdecreases the amount of Tumor Necrosis Factor α (TNFα) produced by acell of the subject, wherein the cell comprises an NMDA glutamatereceptor.
 14. The method of claim 13 wherein the cell is a synovialcell.
 15. The method of claim 13 wherein the neuropathological statecomprises a condition selected from the group consisting of persistentpain, arthritis, ulcerative colitis, inflammatory bowel disease, Crohn'sdisease, pancreatitis, asthma, stroke, brain injury, spinal cord injury,and viral invasion.
 16. The method of claim 13 wherein the compound is atyrosine kinase inhibitor.
 17. The method of claim 16 wherein thetyrosine kinase inhibitor is selected from the group consisting ofGenistein, Lavendustin A, K252a, and a combination thereof. 18-24.(canceled)
 25. A method for treating a neurogenic inflammatory state ina subject, the method comprising: administering to the subject aneffective amount of a compound that decreases the amount of TNFαproduced by a cell of the subject, wherein the cell comprises an NMDAglutamate receptor.
 26. The method of claim 25 wherein the compound is atyrosine kinase inhibitor.
 27. The method of claim 25 wherein the cellis a synovial cell. 28-31. (canceled)